Quantitative measurement of cellular components in a cell by cell assay is usually performed on a cytometer or a hematological analyzer. A blood sample is prepared using specific reagent(s) and procedures and the presence of cellular components is determined using light scatter and fluorescence signals measured on these analyzers. Many physical or chemical factors can affect the ratio of the targeted cellular component present in the cell and the signal output of the measuring analyzer. Without a proper control or reference, it is difficult to obtain an accurate quantitative measurement of the cellular component. A reference control can be included in the measurements of a series of samples, as an external control; or a reference control can be added into each sample to be measured, as an internal control. In the latter case, it is necessary that the analyzer can differentiate between a cellular component from the reference control and the cellular component from the blood cells in the sample being measured.
A reference control can contain a synthetic particle, for example a latex bead covered with a targeted cellular component at a known amount. Other reference controls contain stabilized blood cells, having known amounts of targeted cellular components and being stable for a given period of time at a low non-freezing temperature.
Given the fact that signal variation is not only due to variations inherent to the measuring instrument, but also to variations in the preparation of the samples for the measurement, preferably the particles of a reference control behave similarly to the blood cells of the sample under the condition of sample preparation. In this respect, stabilized blood cells have an advantage over synthetic particles, however, the cellular components or the antigenic properties of the cellular components often deteriorate in the process of preparing the control cells or during their storage at a non-freezing temperature.
Freezing of cells and storage at ultra low temperatures would overcome the problem of degradation over time of the control cells. However, intracellular ice crystals damage the cellular membrane, and special precautions, for example, addition of protective agents such as glycerol or dimethylsulfoxide have to be taken to protect the cells through the freezing and thawing cycle. Even if undamaged, however, the treated cells often exhibit substantial modifications after the freezing and thawing cycle, which affect subsequent measurement. Moreover, the protective agents can also interfere with the assay to be performed.
Therefore, it is desirable that the cells used in the reference control have cell morphology comparable to the blood cells to be assayed and have preserved cellular components and preserved antigens of the cellular components to be measured, and that the cells of the reference control can undergo cycles of freezing and thawing without deterioration of cell morphology and antigens of the cellular components.
Furthermore, although a reference control with a known value of a parameter of interest can be used to correct inter assay variability, it is desirable to have an internal control containing cells in a labeled form to correct for intra assay variability as well as inter assay variability. The labeling of the cells in a reference control allows the measuring instrument to distinguish between the cells in the sample to be measured and the control cells added in the sample and to use these labeled cells as an internal control of the measurement process.
Moreover, in cell by cell assays for measuring intracellular components using antibodies or other molecular probes, it is necessary to permeate the cellular membrane so that the antibodies or other molecular probes can penetrate through the cellular membrane. For such assays, it is desirable to use a reference control containing cells that have their cellular membrane already permeated, which allows large probes such as antibodies to penetrate through.
U.S. Pat. No. 4,777,139 (to Wong et al) teaches a hematology control with red cell components of enhanced stability. Wong et al teach exposing washed red blood cells to an unsaturated aldehyde such as acrolein (propenal) under conditions sufficient to increase the stability of the cells without impairing the ability of a lysing reagent to lyse the cells. After treatment, the treated cells are washed and are suspended in a stabilizing suspension medium. The reference control is stored at low, non-freezing temperature. The membranes of these stabilized cells are not permeated.
The cellular hemoglobin of red blood cells is an important parameter for clinical diagnosis. On most hematology analyzers, total hemoglobin concentration of a blood sample is obtained by lysing red blood cells in a blood sample by a lytic reagent and measuring a chromogen formed by released hemoglobin molecules using spectrophotometric measurement. The mean corpuscular hemoglobin (MCH) of a blood sample is derived from the number of red blood cells (RBC) and total hemoglobin concentration of the blood sample. MCH is an average measurement of all red blood cells, it does not represent hemoglobin content of individual red blood cells.
In contrast, the measurement of cellular hemoglobin on a flow cytometer is a cell by cell measurement, which provides diagnostic information that is not available through MCH. Campbell et al. Cytometry 35, pp 242-248 (1999) have performed flow cytometric analysis of hemoglobin in individual red blood cells using a fluorescent anti-hemoglobin A antibody. Burshteyn et al. (U.S. Patent Application Publication No. 2004/0214243) have performed flow cytometric analysis of hemoglobin using an anti-pan hemoglobin antibody. Since red blood cells have a high concentration of hemoglobin, in order to measure the total cellular hemoglobin using antibodies, a large amount of fluorescently-labeled antibodies are required. Furthermore, there are potential artifacts due to steric hindrance of antibody binding or extinction of fluorescence due to high density of hemoglobin in the cell. Therefore, it is desirable to have a method that enables the measurement of total cellular hemoglobin without relying on the use of antibodies.
Identifying and/or quantifying variants and aberrant forms of hemoglobin are important for clinical diagnosis of various diseases, for example, sickle cell disease, thalassemia and diabetics. The measurement of hemoglobin A1C has been one of most frequently used hemoglobin variant measurement, and is an important clinical measure for diabetic patients.
It is known that about 90% of total hemoglobin is nonglycosylated. The major fraction of nonglycosylated hemoglobin is nonglycosylated HbA, referred to as HbA0. Glycated hemoglobin refers to a series of minor hemoglobin components that are formed via the attachment of various sugars to the hemoglobin molecule. The human erythrocyte is freely permeable to glucose. Within each erythrocyte, glycated hemoglobin is formed at a rate that is directly proportional to the ambient glucose concentration. The reaction of glucose with hemoglobin is nonenzymatic, irreversible and slow, so that only a fraction of the total hemoglobin is glycated during the life span of an erythrocyte (120 days). As a result, the measurement of glycated hemoglobin provides a weighted “moving” average of blood glucose levels that can be used to monitor long-term blood glucose levels, providing an index of the mean blood glucose concentration over the preceding 2 to 3 months. The most important clinical application of this is in the assessment of glycemic control in a diabetic patient.
Hemoglobin A1c (HbA1c) is one specific type of glycated hemoglobin and is the most important hemoglobin species with respect to diabetes. HbA1C is approximately 3 to 6% of the total hemoglobin in nondiabetics, and 20% or greater in diabetes that is poorly controlled (Goldstein, et al., Clin. Chem. 32: B64-B70, 1986). The determination of the concentration of HbA1C is useful in diagnosing and monitoring diabetes mellitus.
Several standard HbA1c assay methods have been developed in the last few decades. One standard method of measuring HbA1c uses ionic-exchange high performance liquid chromatography (HPLC), which separates and analyzes HbA1c and other minor hemoglobin components from hemoglobin HbA0 based upon the differences in charge density. Another chromatography method is boronate affinity chromatography, which uses a gel matrix containing immobilized boronic acid to capture the cis-diol group of glycated hemoglobin. In these methods, a blood sample is lysed first with a lytic reagent, and then formed hemolysate is used in the chromatography analysis.
A further HbA1c assay method is based on immunoturbidimetry. In this method, a blood sample is lysed first with a lytic reagent, and the formed hemolysate is used in two separate measurements. The total hemoglobin concentration is measured by a colorimetric method. HbA1c concentration is measured using the turbidimetric immunoinhibition method, in which HbA1c antibodies in a reagent bind to HbA1c of the sample to form soluble antigen-antibody complexes. Polyhaptens from the reagent then bind with the excess antibodies and the resulting agglutinated complex is measured turbidimetrically. The turbidity of the sample mixture is inversely proportional to the concentration of HbA1c in the sample. The percentage of HbA1c of the sample is calculated using the total hemoglobin and the HbA1c concentration.
These standard HbA1c assay methods are bulk analyses of the average concentration of total hemoglobin and HbA1c percentage of the hemolysate obtained from all lysed red blood cells of a sample, including both mature red blood cells and reticulocytes. This result can only be used as an index of the mean blood glucose concentration over the preceding 2 to 3 months, it does not represent cellular HbA1c percentage of individual red blood cells, and does not provide information of HbA1c percentage between mature red blood cells and immature red blood cells. Therefore, these standard methods do not provide timely information on patient's response to medical treatments.
A number of HbA1c controls are available commercially. Most of these controls are in the form of lyophilized protein powders or hemolyzed liquid solutions, which are used for the above-described methods.
Recently, U.S. Pat. No. 7,361,513 (to Ryan et al.) teaches cellular controls for glycated hemoglobin HbA1c measurement, suitable for use in ion exchange, or affinity chromatography and immunologic detection described above. Ryan et al. teach a method of preparing the reference control, which includes washing a blood sample; removing white blood cells and platelets from red blood cells; fixing the red blood cells with glutaraldehyde; washing and suspending the fixed red blood cells with a stabilizing diluent that contains glucose, sodium fluoride and soybean trypsin inhibitor. Ryan et al. teach this reference control being stable for 300 days at 6° C. and show its utility on several clinical chemistry analyzers that utilize ionic-exchange HPLC, boronate affinity chromatography, or immunoturbidimetry. Using these methods, as described above, the stabilized red blood cells in Ryan et al. are lysed first, then the total hemoglobin and total HbA1c concentration of all red blood cells from the control are measured. Ryan et al do not teach permeating cellular membrane of the fixed red blood cells to render them permeable to antibodies for cell by cell immunoassay on flow cytometer.
Based on the above, it is therefore desirable to provide a reference control containing cellular analogs that have permeated cellular membrane for cell by cell analysis of HbA1c, other hemoglobin variants, or other cellular components on flow cytometer. It is also desirable to have a reference control wherein the total cellular hemoglobin of the cellular analogs can be measured in one step together with a hemoglobin variant. It is further desirable to have a reference control that has labeled cellular analogs so that the analogs can be readily identified and differentiated from blood cells to be measured using commonly available detection devices. Moreover, it is desirable to have a reference control that can be frozen and thawed for long term storage, while maintaining the cellular components of the cellular analogs to be measured, for example, maintaining protein antigenic sites. Furthermore, it is also desirable to have a reference control that is resistant to freeze-thaw treatment without using protective agents that may interfere with the assay to be performed.